Rhouma A, Helali F, Chettaoui M, Hajjouji M, Hajlaoui M R
Laboratoire d'Amélioration et Protection des Ressources Génétiques de l'Olivier, Institut de l'Olivier BP 208 Cité Mahrajène 1082 Tunis, Tunisia.
Laboratoire de Quarantaine Ministère de l'Agriculture.
Plant Dis. 2014 Jan;98(1):158. doi: 10.1094/PDIS-05-13-0542-PDN.
In the spring of 2012 and 2013, symptoms similar to those of fire blight were observed on pear trees (Pyrus communis cv. Alexandrine, Williams) in Tunisia at flowering stages. Disease symptoms appeared in 2012 in the region of Mornag and in the following year extended to the regions of Manouba and Tebourba. More recently, the disease was observed in the regions of Bizerte, Zaghouan, and Beja. The percentages of orchard areas that had infected plants varied from 10 to 40%. Some orchards in Mornag region exhibited more than 75% disease incidence. Symptoms were observed on flowers and young shoots. Blighted blossoms appeared wilted, shriveled, and brown, and dead flowers remained on the stems. Infected shoots wilted rapidly and often formed shepherd's crooks at their tips. Samples of diseased young shoots and flowers were subjected to pathogen isolation and identification. Bacteria were isolated from washed tissues on King's B medium (KB) (1). Colonies with morphology similar to that of Erwinia amylovora were purified by sub-culturing on KB. The strains were first characterized based on morphology and biochemical tests (1). Sixteen strains produced white colonies on KB, were gram-negative, did not produce a fluorescent pigment on KB, did not grow at 35°C, and induced a hypersensitive reaction when infiltrated into tobacco leaves (cv. Xanthi). These strains were identified as E. amylovora by double-antibody sandwich indirect-ELISA and immunofluorescene microscopy using a polyclonal antibody (2) and nested PCR targeting the pEA29 plasmid (3). Pathogenicity was tested using a detached-fruit assay (1). Each strain was inoculated onto three pear fruit (cv. Alexandrine) wounded with a scalpel dipped in a 10 CFU/ml bacterial suspension. The inoculated fruit were incubated at 25°C and 80% relative humidity in plates with sterile 1% agar. Negative controls consisted of fruit wounded with a scalpel dipped in sterile distilled water. Seven days after inoculation, symptoms of discoloration, browning, and production of bacterial ooze appeared at the inoculated points. No symptoms developed on negative controls. Reisolation of bacteria yielded colonies with characteristics of E. amylovora. Purified amplicons from nested PCR were sequenced (KF302525, KF302526) and a BLAST search of the GenBank database revealed 98% homology with E. amylovora strain HF560643.1. References: (1) Anonymous. OEPP/EPPO Bull. 34:159, 2004. (2) M. T. Gorris et al. Acta Hortic. 411:41, 1996. (3) P. Llop et al. Appl. Environ. Microbiol. 66:2071, 2000.
2012年春季和2013年春季，在突尼斯，梨树（西洋梨品种亚历山大、威廉姆斯）花期出现了与火疫病症状相似的症状。2012年，莫尔纳格地区出现病害症状，次年蔓延至马努巴和泰布尔巴地区。最近，比塞大、扎古安和贝贾地区也发现了这种病害。感染植株的果园面积百分比在10%至40%之间。莫尔纳格地区的一些果园发病率超过75%。在花朵和嫩梢上观察到了症状。枯萎的花朵显得枯萎、皱缩且呈褐色，枯死的花朵仍留在茎上。受感染的嫩梢迅速枯萎，顶端常形成曲枝。对患病嫩梢和花朵样本进行了病原菌分离和鉴定。在King's B培养基（KB）（1）上从冲洗过的组织中分离出细菌。通过在KB上继代培养纯化出形态与解淀粉欧文氏菌相似的菌落。首先根据形态和生化试验对菌株进行了鉴定（1）。16个菌株在KB上产生白色菌落，革兰氏阴性，在KB上不产生荧光色素，在35°C下不生长，注入烟草叶片（品种Xanthi）时会引发过敏反应。使用双抗体夹心间接酶联免疫吸附测定法、利用多克隆抗体的免疫荧光显微镜检查法（2）以及针对pEA29质粒的巢式PCR（3）将这些菌株鉴定为解淀粉欧文氏菌。使用离体果实试验检测致病性（1）。将每个菌株接种到用蘸有10 CFU/ml细菌悬液的手术刀划伤的3个梨果实（品种亚历山大）上。接种的果实在含有无菌1%琼脂的平板中于25°C和80%相对湿度下培养。阴性对照为由蘸有无菌蒸馏水的手术刀划伤的果实组成。接种7天后，接种点出现变色、褐变和产生菌脓的症状。阴性对照未出现症状。重新分离出的细菌产生了具有解淀粉欧文氏菌特征的菌落。对巢式PCR纯化的扩增子进行测序（KF302525、KF302526），在GenBank数据库中进行BLAST搜索显示与解淀粉欧文氏菌菌株HF560643.1有98%的同源性。参考文献：（1）匿名。OEPP/EPPO通报。34:159，2004。（2）M. T. 戈里斯等人。园艺学报。411:41，1996。（3）P. 洛普等人。应用与环境微生物学。66:2071，2000。
